Method of cell surface activation and inhibition

ABSTRACT

Disclosed are a peptide and its use in a method of screening test compounds as potential inhibitors of matrix metalloproteinases. The peptide consists of residues Trp 574 to Asp 656 in the TIMP-2 binding site of the C-terminal domain of gelatinase-A as shown by SEQ ID NO:19. The method comprises determining the inhibitory effect of a test compound in a competitive inhibition assay with said peptide in which a Ki/Kd=&gt;1 is deemed an inhibitory effect of said test compound, and in which Ki is the inhibitor constant of said test compound and Kd is the dissociation constant of said peptide.

This is a division of application Ser. No. 08/942,330, filed Sep. 5,1997, now U.S. Pat. No. 6,022,948 which is a continuation of applicationSer. No. 60/026,226, filed Sep. 17, 1996.

FIELD AND OBJECTIVE OF THE INVENTION

This invention relates to methods for cell surface activation andinhibition. More particularly, the invention relates to methods of cellsurface activation and inhibition that involve the interaction of aninhibitor of matrix metalloprotease known as TIMP-2, with the enzyme,gelatinase-A.

Matrix metalloproteases (MMPs) are ubiquitous in human disease anddevelopment. Most processes that involve a certain amount of tissuerepair and damage are believed to be influenced by MMPs such as, forexample, in degradation of type IV collagen that might occur inrheumatoid or osteoarthritis and remodeling of endothelial walls inrestenosis. MMPs are also implicated in various aspects of cancer suchas primary tumor formation, metastasis, and the vascularization oflarger tumors (angiogenesis). It is also known that MMPs are involved inthe conversion of inactive tumor necrosis factor (TNF) precursor intoactive TNF, which in turn is implicated in rheumatoid arthritis, Crohn'sdisease, multiple sclerosis, cachexia and sepsis.

Consequently, the screening for MMP inhibitors as potential drugs is ofsignificant use in the medical and pharmaceutical fields.

MMPs are secreted by mammalian cells as zymogens and upon activationinitiate tissue remodeling by proteolytic degradation of collagens andproteoglycans. Activation of the secreted proenzymes and interactionwith their specific inhibitors, TIMP-1 and TIMP-2, determine the netenzymatic activity in the extracellular space.

TIMP-2 forms a specific complex with the proform of gelatinase-A (GelA)which is mediated by interaction with the C-terminal domain (GelA-CTD)of the enzyme. The amino acid sequence of the 72 kDa GelA is disclosedin Goldberg, U.S. Pat. No. 4,923,818, and its complex with TIMP-2 isdisclosed in Goldberg published European Patent Application, EP 404,750.GelA is a multi-domain protein containing a catalytic domain, a domainwith three type II fibronectin-like repeats, and a C-terminal domain.

Soluble GelA proenzyme is recruited to the cell surface where it isspecifically activated by MT1-MMP, a membrane bound metalloprotease. Thebinding of GelA to cell surface and its subsequent activation is alsomediated by GelA-CTD. Consequently, cell surface activation is inhibitedin the presence of exogenously added excess of TIMP-2 or recombinantGelA-CTD.

It has not been known previously how the MT1-MMP that is inhibited bycomplex with TIMP-2 is able to cleave the GelA propeptide to initiateactivation of the pro-enzyme. Resolution of this question is critical toan understanding of the mechanism by which GelA-CTD interacts withTIMP-2 and MT1-MMP on the cell surface.

BACKGROUND OF THE INVENTION

Literature references on the following background information and onconventional test method and laboratory procedures well known to theordinary person skilled in the art, and other such state-of-the-arttechniques as used herein, are indicated in parentheses, and appended atthe end of the specification.

Secreted metalloprotcases (MMPs) initiate tissue remodeling bydegradation of extraccilular matrix (ECM) macromolecules (reviewed in1-3). Normal physiological processes such as morphogenesis, tissuerepair, and angiogenesis, are dependent upon spatial and temporalregulation of the activity of these enzymes, while malignant cellsexploit these same proteases to promote invasion and metastasis (4-7). Aclear understanding of the mechanisms governing regulation of MMPactivity in extracellular space has remained an elusive goal. TheMT1-MMP/GelA system (8-16) provides a first glimpse at a mechanism bywhich an activity of a soluble MMP, GelA (17), can be spatiallyregulated via its recruitment to the cell surface where the GelAproenzyme is converted into its active form. Transfection of Cos1 cellswith MT1-MMP is sufficient to cause GelA binding to the cell surface andits activation (8,19). The cell surface activation of GelA involves atwo step proteolytic processing of its propeptide. The first cleavage ofthe Asn³⁷-Leu peptide bond is dependant on MT1-MMP (9), a membrane boundmetalloprotease. This cleavage is also dependent on GelA having anintact C-terminal domain since a truncated form of the GCLA proenzymelacking a C-terminal domain can not be activated by membrane boundMT1-MMP (13). Consequently the exogenously added recombinant GelA-CTD)is a competitive inhibitor of Asn³⁷-Leu cleavage (9,10). Finally thisreaction is inhibited in the presence of an excess of inhibitor, TIMP-2,while TIMP-1 has no effect. The consequent cleavage of propeptide isaccomplished via an autoproteolytic, MT1-MMP independent mechanism(9,10,1 8,19) to generate a 62 kDa active GelA with an amino-terminalresidue Tyr⁸¹. These data demonstrate that binding of GelA to the cellsurface via its CTD is a prerequisite for enzyme activation. We havepreviously shown that two closely related proenzymes GelA and B formspecific complexes with TIMP-2 and TIMP-1 respectively (20). Thesecomplexes are also formed via inhibitor interaction with thecarboxyl-end domain of proenzyme (21,22). Thus TIMP-2 and cell surfacebinding activities of GelA-CTD appear to be interrelated. We havepurified activated form of MT1-MMP using affinity chromatographyapproach (9) and demonstrated that it acts as cell surface TIMP-2receptor with Kd=1.65×10⁻⁹M. The MT1-MMP-TIMP-2 complex in turn acts asa receptor for GelA-CTD (Kd=0.42×10⁻⁹M). The data we have presentedsupport the hypothesis that the cell surface binding of GelA-CTD occursvia formation of a tri-molecular complex of activatedMT1-MMP/TIMP-2/pro-GelA that promotes pro-GelA activation. This model,however, does not satisfactory resolve the GelA activation mechanism forthe following reasons. The inhibitor TIMP-2 consists of two domains. Theamino-terminal, inhibitory domain interacts with the active center ofMMPs to form an inhibitory complex (23,24). The C-terminal domain bindsto GelA-CTD. Thus the inhibitory complex of TIMP-2 with activatedMT1-MMP can leave the C-terminal domain of the inhibitor exposed andavailable for interaction with GelA-CTD. In fact we have reported ananalogous tri-molecular complex between GelB, TIMP-1 and activatedinterstitial collagens (22) where the collagens component of the complexwas inhibited. Moreover the specific inhibition of soluble form ofMT1-MMP by TIMP-2 has been recently demonstrated (25,26). Thus, themodel of cell surface GelA activation that requires assembly of theMT1-MMP/TIMP-2/pro-GelA complex leaves unanswered the question of howthe MT1-MMP inhibited by TIMP-2 is able to cleave the Asn³⁷-Leu peptidebond to initiate activation of the pro-enzyme. An answer to thisquestion demands a better understanding of the mechanism by whichGelA-CTD interacts with TIMP-2 and MT1-MMP on the cell surface. We haverecently reported the high resolution crystal structure of GelA-CTD(27). Here we report the results of extensive alanine scanningmutagenesis of solvent exposed GelA-CTD amino-acid residues and, usingthe coordinates of the GelA-CTD structure, define a TIMP-2 binding siteon the surface of this domain. By comparison of the TIMP-2 binding siteto the same regions in related MMP structures, we characterizestructural features required for general TIMP binding and thespecificity of TIMP-2- GelA-CTD interaction. We also report analysis ofGelA activation inhibition activity of GelA-CTD mutants relative to thatof wild type.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, methods of cell surfaceactivation and inhibition are provided which are useful for thescreening of MMP inhibitors that are potentially useful for thetreatment of diseases that involve tissue repair and damage and otherdiseases in which MMPs are implicated.

Critical to the methods of the invention is the discovery of a uniqueportion of the TIMP-2 binding site on the surface of the GelA-CTDdomain, which has been determined herein to be the very strongly bindingresidue Asp⁶⁵⁶. This critical TIMP-2 binding site can also include otherresidues in the GelA-CTD domain with which Asp⁶⁵⁶ forms a contiguoussurface, namely the less strongly binding residues Gly⁶⁵¹, Phe⁶⁵⁰, andTyr⁶³⁶.

In accordance with another embodiment of the invention, the TIMP-2binding site includes the foregoing four residues and additionally thevery strongly binding residues Asp⁶¹⁵, Lys⁶⁴⁶, Lys⁵⁷⁶, Trp⁵⁷⁴, andArg⁵⁹⁰, and the less strongly binding residues Lys⁵⁷⁹, Lys⁶⁰⁴ andAsn⁶¹¹. The effect of these residues on the TIMP-2 binding of GelA-CTDhas been confirmed by mutagenesis.

Point mutations can be made at these residues in the TIMP-2 binding siteto impact the TIMP-2 binding to GelA-CTD, e.g., to inhibit or retard thebinding, and thereby provide a unique screening method.

Identification of this TIMP-2 binding site provides a useful target forthe screening of MMP inhibitors and for the prognosis and treatment ofdiseases in which MMPs are implicated. Compounds which are structured tocompetitively inhibit cell surface activation can be candidate MMPinhibitors.

DETAILED DESCRIPTION OF THE INVENTION

The following description of the invention taken in conjunction with theaccompanying drawings is provided to further illustrate the inventionand preferred embodiments in greater detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

Figure Legends.

FIG. 1A and B. Space-filling model (A, 34) and ribbon diagram (B, 35) ofGel A-Ctd showing residues which interact with TIMP-2. A) The residueswhich are thought to directly interact with TIMP-2 are shown colored andlabeled. Residues which showed between a 2- to 100-fold loss in TIMP-2binding when mutated to alanine are colored cyan, while those whichshowed over 100-fold loss in TIMP-2 binding when mutated to alanine arecolored dark blue. TBS-1 and TBS-2 regions are indicated by the dashedmagenta boxes which cover their respective regions. B) A ribbon diagramof Gel A-Ctd shows the canonical β-propeller fold. Each blade of GelA-Ctd is labeled with a roman numeral. The Ca2+ ion is shown in redalong the central axis of symmetry. The disulfide bond connecting bladesI and IV is shown as are the N- and C- termini. Residues which arethought to directly interact with TIMP-2 are shown in magenta and arelabeled. All the residues lie on blade III, blade IV or the loopconnecting the two blades.

FIG. 2. Competition Assay of GelA-Ctd Binding to TIMP-2. One hundred μLof solution containing 1.7×10⁻⁹M of ¹²⁵ I-labeled WT GelA-Ctd (10⁸cpm/μg) and unlabeled purified recombinant WT or mutant of the GelA-Ctdat the indicated concentrations were incubated in TIMP-2 (50 ng) coatedwells of microtiter plates and washed as described in Methods. Boundradioactivity was determined by counting individual wells in a gammacounter. After background substraction the CPM retained in the wells wasnormalized to 1.00 and plotted versus the concentration of unlabeled,competing Gel A-Ctd for wild type (⋄, Ki/Kd=1) or mutants Gly⁶⁵¹ (X,Ki/Kd=3); Lys⁵⁷⁹ (□, Ki/Kd=6); Lys⁶⁰⁴ (Δ, Ki/Kd=25); and Asp⁶¹⁵ (∘,Ki/Kd=300) as indicated in the figure. The values for wild type selfcompetition are the mean of 8 separate experiments and the error barsrepresent the standard error. The mutant values are the average of twodeterminations. Computer generated theoretical curves were fitted to thedata and the apparent Kd for WT and Ki for mutants were determined fromthe fit as shown in the figure by each solid curve.

FIG. 3. Molecular surface of Gel A-Ctd showing the TIMP-2 binding site.A molecular surface of Gel A-Ctd was calculated and displayed usingGRASP. The TIMP-2 binding site is colored magenta. The yellow labelsdenote some of the TIMP-2 binding residues and their position withrespect to the surface. The boundary residues described in the paper areshown in green and are seen to surround the TIMP-2 binding site.

FIG. 4. Comparison of molecular surface and electrostatic potential atsurface of Gel A-Ctd and ClI-Ctd). The molecular surface of both (4A)Gel A-Ctd and (4B) ClI-Ctd are displayed. Electrostatic potential foreach was calculated and displayed with positive potential shown in blueand negative potential in red. The dashed magenta line conforms to theapproximate TIMP-2 binding site described in the text. TBS-1 and TBS-2are shown and conform to the same regions described in FIG. 1. The TBS-1region of Gel A-Ctd displays a lot of positive potential, where asClI-Ctd has much less positive potential and has a significant amount ofnegative potential across the TBS-1 binding interface. The TBS-2 regionsof both molecules show differing potentials as well. The molecularsurfaces of both molecules suggests that they would presentsignificantly different van der Waal contact surfaces.

FIG. 5. Sequence alignment of β-propeller blades III and IV fromC-terminal domains of MMP family members. The amino acid sequence ofGelA containing all the residues defining the TIMP-2 binding site wasaligned with other MMPs. Sequences which are found in blade III or bladeIV are beneath the underlined regions. GelA residues which are part ofthe TIMP-2 binding site and corresponding residues from other enzymesare bolded. Those residues constituting the TBS-1 region (see text) arebolded and underlined, while the remaining residues are part of TBS-2are merely bolded. An ‘*’ marks residues whose effect on TIMP-2 bindingof GelA-CTD were confirmed by mutagenesis. The residues defining theTIMP-2 binding site are thus shown to comprise the peptide from thefirst * to the last * in the GelA-CTD, namely, Trp⁵⁷⁴ to Asp⁶⁵.

FIG. 6. Inhibition of membrane dependent activation of GelA by GelA-CTDmutants. The 15 ng of purified GelA were incubated in 25 mM HEPES-KOHbuffer, pH 7.5, containing 0.1 mM CaCl₂ with 20 μg of plasma membraneprotein from HT1080 cells for 2 h at 370° C. in the presence ofincreasing concentration (1-6) of recombinant GelA-CTD WT or mutants #28(Asp⁵⁶⁹), #31 (Lys⁵⁷⁹), #39 (Lys⁶⁰⁴), #41 (Asp⁶¹⁵), #229 (Asp⁵⁷⁶), #234(Arg⁵⁹⁰), #247 (Lys⁶⁴⁶), #250 (Trp⁵⁷⁴), #252 (Tyr⁶³⁶), #255 (Phe⁶⁵⁰),#257 (Gly⁶⁵¹), #258 (Asp⁶⁵⁶), #259 (Asn⁶¹¹) as indicated in each panel.The results of activation reaction were analyzed on zymogram asdescribed previously (9,10). The images of resulting zymograms wereacquired using flat bed scanner and converted to a negative.

The colored areas in FIG. 1A and FIG. 3 are shown in black and whitecopies as follows:

FIG. 1A—Residues shown in dark blue are Asp⁶⁵⁶, Asp⁶¹⁵, Lys⁶⁴⁶, Lys⁵⁷⁶,Trp⁵⁷⁴ and Arg⁵⁹⁰. Residues shown in cyan are Gly⁶⁵¹, Phe⁶⁵⁰, Tyr⁶³⁶,Asn⁶¹¹, Lys⁵⁷⁹ and Lys⁶⁰⁴.

FIG. 3—Boundary residues shown in the green colored area are Lys⁶⁴⁹,Gln⁶⁴¹Lys⁵⁷⁸, Lys⁶³³, Asp⁶⁰⁸ and Asp⁶¹⁸. The red colored TIMP-2 bindingsite shows residues Asp⁶⁵⁶, Phe⁶⁵⁰, Tyr⁶³⁶, Asp⁶¹⁵, Asn⁶¹¹, Lys⁶⁴⁶,Lys⁵⁷⁶, Trp⁵⁷⁴ and Lys⁶⁰⁴.

In order to further illustrate the invention, the following detailedexamples were carried out although it will be understood that theinvention is not limited to these examples or the details describedtherein.

EXAMPLES Materials and Methods

Cell Culture.

HT1080 fibrosarcoma cells were grown in monolayer culture in RPMI 1640media supplemented with 4% fetal calf serum and 2 mM glutamine in thepresence of 5% CO₂ and treated with 12-O-tetradecanoyl-phorbol acetate(TPA) (50 ng/ml for 16 h). Isolation of plasma membranes from HT1080cells was performed using discontinuous sucrose gradient as described(9,10).

Enzyme Purification.

The GelA expression plasmid p6R72hyg was transfected into E1A-expressingp2AHT2a cells and GelA was purified from conditioned medium of stablytransfected cell line p2AHT7212A as described (9,10)

Expression and Purification of TIM P-2.

Recombinant TIMP-2 was expressed in p2AHT2a cells transfected withTIMP-2 cDNA in the p6Rhyg expression vector and purified from serum freeconditioned media of p2AHT2aT2 cells as described earlier (9,10) usingReactive Red-120-Agarose (Sigma, R-0503), Q-Sepharose (Pharmacia#17-0510-01), CM-Sepharose CL-6B (Sigma #CCL-6B-100) and RP-HPLC columnchromatography.

Expression and Purification of the FLAG GelA-CTD Fusion Protein.

Expression vector pFLAG72CT was constructed by cloning a fragment fromGelA cDNA (17) coding for Leu⁴⁴⁴-Cys⁶⁶⁰ into E.Coli secretion vectorpFlag1 (IBI Inc.). The resulting vector coding for the fusion proteinFLAG-CT was transfected into an E.coli TOPP5 host (Stratagene). Proteinwas purified from a periplasmic fraction by chromatography on ReactiveRed-120-Agarose (Sigma, R-0503) and M1 anti-flag antibody affinitycolumn as described previously (9,10,27). Each of the 50 mutants andwild type GelA CT were purified using this procedure.

Mutagenesis of the FLAG GelA-CTD Fusion Protein.

Expression vector pFLAG72CT was mutagenized directly using PCR mediatedsite directed mutagenesis. A pair of anti-parallel 33 base pair longprimers was synthesized for each mutant. These primers containing adesired mutation were used in a pair of PCR reactions with either of twoprimers flanking the coding sequence. Both resulting PCR productscontained mutation. They were mixed, melted and annealed to generate apartial heteroduplex encompassing the whole coding sequence. The latterserved as a template in a third PCR reaction primed by both of theflanking primers. Each of the resulting PCR products was cloned backinto the pFLAG72CT expression vector and subjected to a sequenceanalysis to confirm the presence of mutation. All resulting mutantproteins were purified and assayed for TIMP-2 binding as describedbelow. The sequence of mutants that had negative effect on TIMP-2binding was verified by sequencing of the entire coding region toexclude the appearance of secondary, PCR generated, mutations. Secondarymutations, when present, were separated from the desired mutant byeither a second round of PCR or using restriction enzyme mediatedsubcloning.

TIMP-2 binding of the FLAG-GelA-CTD Fusion Protein.

The TIMP-2 binding and competition assays were performed in 96 wellmodular plates (Costar). TIMP-2 coated plates were prepared by additionof 100 μl of loading buffer (20 mM Tris HCl, pH 9) containing 50 ng ofpurified TIMP-2 to each well and incubated for 1 h at RT. This solutionwas replaced with 200 μl of blocking buffer (0.5% BSA and 0.02% Brij inPBS, pH 7.2) and incubated ON at 4° C. For binding experimentsincreasing concentrations of competing cold ligand in 100 μl of bindingbuffer (1 mg/ml BSA and 0.01% Brij in PBS) were added to TIMP-2 or BSA(control) coated wells and incubated for 30 min prior to addition of10⁻⁹M of ¹²⁵I-GelA-CTD (between 6,5×10⁷ and 1×10⁸ dpm/μg). Incubationcontinued for 1 h, after which plates were washed 5 times with BindingBuffer and each well was counted to determine retained radioactivity.

Activation of the GelA Proenzyme.

Between 15-50 ng of the GelA proenzyme was used for activation withplasma membranes (1-4 μg of plasma membrane protein) in 10 μl finalvolume of 25 mM HEPES-KOH buffer, pH 7.5, containing 0.1 mM CaCl₂. Thereaction was incubated at 37° C. for 120 min, terminated by addition ofthe sample buffer and subjected to gelatin zymogram analysis asdescribed (9,10).

Protein Structure Analysis.

Residues whose mutation to alanine caused a loss in TIMP-2 binding weredivided into those that most likely directly interact with TIMP-2 andthose whose effect on TIMP-2 binding arc most likely a result ofindirect structural perturbations based on a detailed examination of theenvironment of each of the mutant. The set of residues which interactwith TIMP-2 are all confined to a single, contiguous surface of GelA-CTDwhich is divided into two adjacent regions, TBS1 and TBS2. Usingboundary residues which are near the TIMP-2 binding residues but whosemutation to alanine had no effect on TIMP-2 binding permitted us todefined the TIMP-2 binding site as a molecular surface that includesresidues not mutated in the analysis.

GelA-CTD and the C-terminal domain of interstitial collagens (ClI-Ctd)were aligned along their respective C_(α) atoms. The two structuresaligned with an average root mean square difference in C_(α) position of3.7 Å and were visualized using the graphics program O (28). The modelof GelB-CTD was constructed using the modeling software, Sybyl (version6.2, Tripos Associates, St. Louis, Mo.). The GelA-CTD structure providedthe basic template for the structure and the coordinates of the C_(α)atoms were preserved in regions of sequence identity. In these regions,the conformation of the sidechains were preserved as well. In regionswith no sequence identity, the C_(α) positions were held constant butthe side chain conformation was chosen from a rotamer library set.Steric clashes due to the insertion of GelB residues were relieved bymoving either the neighboring atoms (whether they be sidechains orbackbone atoms) or by moving the C_(α) position of the substitutedresidue. Regions requiring the insertion or deletion of residues in thesequence only occurred along loops or turns and were modeled by choosinga turn or loop from the Brookhaven protein data bank that had a similarsequence and made the fewest van der Waal contacts with nearby atoms.Finally, the model was completed by minimizing van der Waal contactsover the entire structure. The final GelB-CTD model was aligned withGelA-CTD along their respective C_(α) atoms.

Results.

Description of GelA-CTD Structure

The GelA-CITD coordinates are from a high resolution crystal structure(resolution=2.15 Å) with a low R-factor (18.8%) and low averagecoordinate crror (<0.25 Å) (27), so the positions of the backbone andsidechain atoms are well determined. The structure includes all residuesbetween Leu⁴⁶¹ and Cys⁶⁶⁰ where the only residues with poorly definedpositions are Glu⁵²⁹ and Glu⁵³⁰. The overall structure of GelA-CTD isbest described as a four-bladed β-propeller (FIG. 1). The four ‘blades’are each composed of four strands of anti-parallel P-sheet. The β-sheetdomains are twisted making the fourth, outer most strand form nearly an80° angle with the inner most strand. Each blade is arrayed about acentral pseudo four-fold axis so that a 90° rotation about the axispositions one blade on top of another. A channel formed by the fourblades, parallel to the rotation axis, contains a Ca²⁺ ion, a Na⁺Cl ionpair and a number of stably bound water molecules. The inner moststrands of each blade are all parallel and the Ca²⁺ ion protrudes fromthe N-terminal end of the channel. The regions between the four bladesare composed of hydrophobic residues (primarily Phe, Tyr and Trp) whichare large enough to contact one another across such a wide interface.Connecting loops lay across the hydrophobic interface and connectadjacent blades. Blade IV is connected covalently to blade I via adisulfide bond between Cys⁴⁶⁹ and Cys⁶⁶⁰.

Identification of the TIMP-2 Binding Site in the GelA-CTD by AlanineScanning Mutagenesis.

Alanine scanning mutagenesis of solvent exposed amino-acid residues ofGelA-CTD) was used to define its molecular surface that interacts withTIMP-2. The results were interpreted by examination of the location andenvironment of each point mutant in the crystal structure of GelA-CTD(FIG. 1, 27), so that only residues of GelA-CTD which can directlyinteract with TIMP-2 are identified. Expression vector pFLAG72CT wasmutagenized directly using PCR mediated site directed mutagenesis asdescribed in Methods. All fifty resulting mutant proteins were purifiedas described previously (9,10) and assayed for TIMP-2 binding asdescribed in Methods. The sequence of mutants that had negative effecton TIMP-2 binding was verified by sequencing of the entire coding regionto exclude the appearance of secondary, PCR generated, mutations. Toquantitate the TIMP-2 binding affinity of the different GelA-CTD mutantsrelative to wild type (WT) GelA-CIT we developed TIMP-2 binding andcompetition assay in 96 well modular plates. For binding experiments asolutions of 10⁻⁹M of ¹²⁵I-GelA-CTD containing increasing concentrationsof competing cold ligand were added to TIMP-2 or BSA (control) coatedwells and retained radioactivity was determined by counting individualwells as described in Methods. The apparent Ki for each mutant wasdetermined by a fit of computer generated series of curves to the datafrom the competition assay. A 25% variation in apparent Ki thusdetermined produced curves which were clearly less representative of thedata. An example of the results of this analysis for WT GelA-CTD andfour mutants are shown in FIG. 2. The mutants presented in FIG. 2 werechosen to illustrate the range of variation encountered. All the mutantsthat had an effect on TIMP-2 binding (Ki/Kd>1) are summarized inTable 1. Substitution of Ala for one of the following amino acidresidues Lys⁴⁷⁰, Arg⁴⁸², Arg⁴⁹¹, Arg⁴⁹⁵, Asp⁵⁰¹, Glu⁵¹⁵, GlU⁵¹⁸, Lys⁵¹⁹,Glu⁵²⁹, Lys⁵³¹, Glu⁵³⁹, Glu⁵⁴⁹, Arg 550, Asp⁵⁶⁴, Arg⁵⁶⁷, Lys⁵⁷⁸, Asp⁵⁸⁶,Lys⁵⁹⁶, Asp⁶⁰⁸, Asp⁶¹⁸, Hys⁶²⁸, Lys⁶³³, Lys⁶³⁹, Glu⁶⁴¹, Lys⁶⁴⁹, Leu⁶³⁸,Gln⁶⁴³, and Leu⁵⁴⁸ did not affect the binding affinity of GelA-CTD toTIMP-2 (Kd=Ki) in this assay. Single replacement of Lys⁵¹⁹ with Arg,Ala⁴⁷⁹ with Thr, or Leu⁵⁴⁸ with Arg also had no effect.

Localization of TIMP-2 Binding Residues on GelA-CTD.

Among all the point mutants of GelA-CTD which show a loss in binding,only Asp⁵⁶⁹ is not considered part of the TIMP-2 binding surface. Theremaining mutants all lie within two adjacent areas of the GelA-CTDshown as TIMP-2 Binding Surface-1 (TBS-1) and TIMP-2 Binding Surface-2(TBS-2) in FIG. 1. The TIMP-2 binding site of GelA-CTD is divided intotwo regions in order to facilitate discussion of the different featuresseen in this broad binding site and to simplify comparison of theseregions on related proteins. There is no physical basis for dividing thebinding site into two regions, but we do so in order to discussdifferent features seen in the TIMP-2 binding site. TBS-1 is formedbetween blades III and IV and includes a non-polar interface composed oflarge aromatic residues (contacting Trp⁵⁷⁴) which pack between the twoadjacent blades and form a small, hydrophobic cavity. Surrounding thisnon-polar part of TBS-1 are a number of positively charged residueswhich are contributed mostly from the second (Lys⁵⁷⁶, Lys⁵⁷⁹), third(Arg⁵⁹⁰), and fourth (Lys⁶⁰⁴) strands of blade III as well as Lys⁶⁴⁶which is on a large turn made between the third and fourth strands ofblade IV. The non-polar cavity is bounded by a looping strand which liesacross the cavity and connects blades III and IV. This loop region,which contains Asn⁶¹¹, is considered part of TBS-1 but is adjacent toTBS-2 and forms part of the putative TIMP-2 binding surface of GelA-CTD.TBS-2 contains residues required for TIMP-2 binding that are mostlylocated on blade IV. PhC⁶⁵⁰ and Gly⁶⁵¹ are located on the fourth strandof blade IV. Tyr⁶³⁶ comes from the third strand of blade IV but forms anadjacent surface with Phe⁶⁵⁰ and Gly⁶⁵¹. Asp⁶⁵⁶ is located on a singleα-helical turn at the end of blade IV. Asp⁶¹⁵ is part of the loopsection connecting blades III and IV, but is positioned adjacent toTyr⁶³⁶. Together, TBS-1 and TBS-2 make up the entire putative TIMP-2binding surface of GelA-CTD. From FIG. 1, it can be seen that residueswhose mutation caused at least 100-fold loss in TIMP-2 binding arepredominantly found in TBS-1 in and about the cavity. Asp⁶¹⁵ is the onlyresidue from TBS-2 which showed more 100 fold loss in TIMP-2 bindingwhen mutated to alanine.

In modeling a TIMP-2 binding surface of GelA-CTD, it is possible to alsomake use of point mutations which had no effect on TIMP-2 binding. Someresidues on GelA-CTD near or adjacent to the putative binding region didnot impact TIMP-2 binding when mutated to alanine.

These mutants are considered boundary residues because they help definethe outer limits of the TIMP-2 binding surface. They include Lys⁵⁷⁸,Asp⁵⁸⁶, Asp⁶⁰⁸, Asp⁶l⁸, Lys⁶³³, Lys⁶³⁹, Glu⁶⁴¹, Gin⁶⁴³, and Lys⁶⁴⁹.While the list is not an exhaustive one and does not completely surroundthe site, it is a considerable number, and as seen in FIG. 1, theycontribute greatly to determining the shape of the TIMP-2 bindingsurface on GelA-CTD.

The effects of point mutations on GelA-CTD) binding of TIMP-2 can becharacterized as ‘direct’ or ‘indirect’. Point mutations with directeffect presumably show a loss in binding due to direct interaction withTIMP-2 since in the crystal structure these residues are almost entirelysolvent exposed making no significant van der Waals contact, saltbridges or hydrogen bonds with nearby sidechain or backbone atoms. Thoseclassed as ‘indirect’ are point mutants of residues which are involvedin such interactions with neighboring atoms. The effect of these mutantson TIMP-2 binding may be either a result of loss of direct interactionwith TIMP-2 or due to a perturbation of the local structure as a resultof the point mutation which ‘indirectly’ causes a loss in TIMP-2binding. Most of the point mutants which have an effect on TIMP-2binding are classed as ‘direct’ including mutants of Lys⁵⁷⁶, Lys⁵⁷⁹,Arg⁵⁹⁰, Lys⁶⁰⁴, Asn⁶¹¹, Asp⁶¹⁵, Lys⁶⁴⁶, and Phe⁶⁵⁰ (see Table 1). Tyr⁶³⁶may also be considered direct in that most of the ring including thehydroxyl group is solvent exposed and the van der Waals interactions ofits C_(δ)1 and C_(ε)1 atoms are not likely to significantly perturblocal geometry. The residues classed as ‘indirect’ are Asp569, Trp5⁷⁴,Gly⁶⁵¹, and Asp⁶⁵⁶. The residues classed as ‘indirect’ are ASp⁵⁶⁹,Trp⁵⁷⁴, Gly⁶⁵¹, and Asp⁶⁵⁶.

The entire TIMP-2 binding site shown in FIG. 3 represents a surface areaof 1027 Å². The interior of the surface is defined by residues whosepoint mutants show a loss in TIMP-2 binding. The boundary of the surfaceis defined by the outermost residues which show an effect on TIMP-2binding and by the boundary residues described above. In order to createthe entire surface other residues for which mutations were not madeneeded to be included as part of the binding surface. These residueswere selected by the criteria that they could have no atoms outside theboundary of the binding surface and must have surface accessible atomswithin the interior of the surface. The non-mutated residues included aspart of the TIMP-2 binding surface are residues Asn⁵⁷⁷, Tyr⁵⁸¹, Phe⁵⁸⁸,Ala⁶⁰⁹, Trp⁶¹⁰, Ala⁶¹², Ilc⁶¹³, Pro⁶¹⁴, Leu⁶⁴⁵, and Val⁶⁴⁸ as well asthe C_(ζ) and C_(ε)1 ring carbons of Phe⁶⁰². All the aromatic residuesof this group as well as Leu⁶⁴⁵ contribute to form the non-polar cavityin TBS-1. Ala⁶¹², IlC⁶¹³, and Pro⁶¹⁴ are on the loop connecting bladesIII and IV. Ilc⁶¹³ is unique in that only its backbone atoms are surfaceaccessible. Val⁶⁴⁸ makes up part of the van der Waal contact surface ofTBS-2. The hole in the binding surface is present because Leu⁶³⁸ (whichis in a small depression between Phe⁶⁵⁰ and Tyr⁶³⁶) did not show a lossin TIMP-2 binding when mutated to alanine. Thus, the C_(γ), C_(δ)1 andC_(δ)2 atoms of Leu⁶³⁸ are not considered part of the TIMP-2 bindingsurface. Rationalizing the effects of these mutations on TIMP-2 bindingrequires a broader description of the structural environment of theseresidues.

Structural Analysis of ‘Indirect’ Mutants

As discussed above, a some of the residues included in the TIMP-2binding site of GelA-CTD may be classed as ‘indirect’ mutants includingAsp⁵⁶⁹, Trp⁵⁷⁴, Gly⁶⁵¹, and Asp⁶⁵⁶. Here, we rationalize the effects ofmutating them to alanine on TIMP-2 binding given the fact that theseresidues also interact with other portions of the GelA-CIT) molecule.The O_(δ1 of Asp) ⁵⁶⁹ forms a hydrogen bond with a backbone amide protonof Gly⁵⁸⁵ which is located on a tight turn formed between the second andthird strands of blade III. The Asp⁵⁶⁹->Ala mutation causes only a smallreduction in TIMP-2 binding. Strand three of blade III contains aresidue, Arg⁵⁹⁰, whose mutation to alanine shows a large loss in binding(>100-fold) and directly interacts with TIMP-2. Also, Asp569 is awayfrom the contiguous binding surface formed by the other point mutantswhich affect TIMP-2 binding. While it is possible that TIMP-2 directlyinteracts with Asp⁵⁶⁹, the effect of the Asp⁵⁶⁹->Ala mutation is mostlikely mediated by alteration of the position of Arg⁵⁹⁰ as a result ofthe loss of an important structural H-bond with Gly⁵⁸⁵ which constrainsthe conformation of the turn.

Trp⁵⁷⁴ is one of a number of hydrophobic residues forming the pocketbetween blades III and IV. It makes van der Waals contact with a numberof atoms from neighboring sidechains including Tyr⁵⁸¹ and Trp⁶¹⁰. Sinceonly the C_(ζ)3 and C_(η)2 atoms of Trp⁵⁷⁴ are surface accessible, thelarge loss in binding of the Trp⁵⁷⁴->Ala mutant is most likely not dueto a loss in the interaction of these atoms with TIMP-2 but to arearrangement of neighboring residues as a result of the mutation. Themost reasonable interpretation of the effect of the mutation is thatTIMP-2 makes van der Waals contact with this pocket upon bindingGelA-CTD and that Trp⁵⁷⁴->Ala disrupts TIMP-2 binding by altering thevan der Waal surface presented by GelA-CTD. So while the effect of theTrp⁵⁷⁴->Ala mutation is ‘indirect’, it is suggestive of direct bindingof TIMP-2 to surface atoms in the pocket.

The Gly⁶⁵¹->Ala mutation has a moderate effect on TIMP-2 binding. Sincealanine, by virtue of its C_(α), is sterically restricted in itsallowable φ, ψ angles, it is possible that the effect on TIMP-2 bindingof the Gly⁶⁵¹->Ala mutation is the result of alteration in the proteinbackbone. Alanine is more energetically restricted than glycine in theφ, ψ conformations it may adopt. However, since Gly⁶⁵¹ is found in awell formed β-sheet of the outer most β-strand of blade IV and adoptsphi psi angles (φ=−157.9, ψ=−174.8) commensurate with an anti-parallelβ-stand conformation, alanine is likely to adopt the same conformationat the site. Thus, loss in TIMP-2 binding for the alanine mutant is dueto the addition of the C_(β) atom on residue 651 which, byapproximation, blocks interaction of TIMP-2 with the C_(α) of 651. Thedouble mutant, Glu⁶⁵⁴->Ala/Gly⁶⁵¹->Arg shows>100-fold loss in TIMP-2binding. Glu⁶⁴¹ is entirely solvent exposed and does not interact withneighboring atoms, and the single mutant, Glu⁶⁴¹->Ala shows no effect onTIMP-2 binding. Since Glu⁶⁴¹ is entirely solvent exposed does not appearto interact with any neighboring atoms, the effects of the double mutantare not considered to be a result of cooperativity. Thus, the effect ofthe double mutant is due exclusively to the Gly⁶⁵¹->Arg mutation.Presumably, the Gly⁶⁵¹->Arg mutant covers a nearby surface on GelA-CTDupon which TIMP-2 normally binds. Since arginine is much larger thanalanine and is also charged, it is not surprising that it had a muchmore dramatic effect on TIMP-2 binding than alanine. The fact that bothGly⁶⁵¹->Ala and Gly⁶⁵¹->Arg reduce TIMP-2 binding suggests that TIMP-2contacts GelA-CTD at the C_(α) of Gly⁶⁵¹ as well as surface residuesnear Gly⁶⁵¹. Asp⁶⁵⁶ is solvent exposed and forms an H-bond with thehydroxyl group of Tyr⁶³⁷. The effect on TIMP-2 binding of theAsp⁶⁵⁶->Ala mutation may be a result of perturbation of the orientationof Tyr⁶³⁷ by loss of this H-bond. While it is possible that TIMP-2interacts with only Tyr⁶³⁷, the simplest interpretation of the effect ofthe mutation is that Asp⁶⁵⁶ directly interacts with TIMP-2. Thisconclusion would partially explain the large loss in TIMP-2 binding ofthe Gly⁶⁵¹->Arg mutation which puts a positive charge near Asp⁶⁵⁶. Also,Asp⁶⁵⁶ forms a contiguous surface with Gly⁶⁵¹, Phe⁶⁵⁰, and Tyr⁶³⁶ (otherTIMP-2 binding residues). TIMP-2 may interact with Tyr⁶³⁷ but thatresidue was not mutated in the study so it cannot explicitly beconsidered as part of the TIMP-2 binding surface of GelA-CTD.

Comparison of GelA-CTD and Interstitial Collagenase.

Having defined a TIMP-2 binding site on the surface of GelA-CTD, it isinstructive to compare the known structure of the C-terminal domain ofinterstitial collagenase (ClI-Ctd) (29) which does not bind TIMP-2 toidentify which structural features of the TIMP-2 binding site are sharedand which are divergent. It was surprising to find that many of thepositively charged residues are conserved both in terms of sequence andstructure. Lys⁵⁷⁹, Arg⁵⁹⁰, and Lys⁶⁰⁴ of GelA-CTD are conserved inClI-Ctd and adopt similar conformations in the structures (FIGS. 3 and4). Furthermore, Lys⁶⁴⁶ in GelA-CTD aligns with Arg⁴⁵³ in ClI-Ctd, sowhile the sequence is not identical, charge is conserved and theresidues overlay well when their C_(α) atoms are aligned. Point mutantsof Arg⁵⁹⁰ and Lys⁶⁴⁶ all show at least 100-fold loss in TIMP-2 bindingin GelA-CTD. Lys⁵⁷⁶, which also shows over a 100-fold loss in TIMP-2binding when mutated to alanine, is not conserved in ClI-Ctd where itbecomes a negatively charged Asp residue. It is interesting to note thatsome of the charged residues, like Arg590 and Lys646, which seem to makea large contribution to TIMP-2 binding are also conserved in a ClI-Ctdwhich does not bind TIMP-2. Clearly, other features of GelA-Ctd, whichare not found in ClI-Ctd, must be identified to account for its TIMP-2binding properties.

Further examination of the aligned structures reveals that the non-polarcavity in GelA-CTD is covered by a number of negatively charged residuesin ClI-Ctd. Trp⁵⁷⁴, Lys⁵⁷⁶, and Ala⁶⁰⁹ of GelA-CTD align with negativelycharged residues of ClI-Ctd. In ClI-Ctd, Asp³⁸⁵ is on the periphery ofthe pocket; Glu³⁸³ protrudes from the pocket and Glu⁴¹⁸ extends over thepocket. The effect of these negative charges on TIMP-2 binding is stillnot known but their negative potential could shield the nearby positivecharges from TIMP-2. Alternatively, if TIMP-2 does make van der Waalscontacts with the non-polar cavity of GelA-CTD, the effect of all thecharged groups in the cavity would be to block this interaction and infact bury the negative charges inside the TIMP-2/GelA-CTD bindinginterface. The negative potential in the cavity of ClI-Ctd is partiallyreduced by the presence of Lys⁴⁵² which is Leu⁶⁴⁵ in GelA-CTD. Leu⁶⁴⁵ isa non-polar residue which points into the hydrophobic cavity of TBS-1.FIG. 3 shows the charge potentials of both GelA-CTD and ClI-Ctd ascalculated and displayed by GRASP. Comparison of the TBS-1 regions ofGelA-CTD) and ClI-Ctd suggests qualitatively that the pockets formedpresent different accessible surfaces. Some residues in the pocket areconserved, notable exceptions are Trp⁶¹⁰ and Phe⁵⁸⁸ of GelA-CTD). Otherdifferences are seen in the loop connecting blades III and IV. Here,ASp⁶¹⁵ becomes isosteric, but uncharged Asn⁴²⁴ in ClI-Ctd, while Asn⁶¹¹,Ala⁶¹², Pro⁶¹⁴ of GelA-CTD are changed to other residues in ClI-Ctd.Only Ile⁶²³ is conserved but this residue has only backbone atoms whichare surface accessible in the structure. Clearly, GelA-CTD and ClI-Ctdwould present a very different charge distribution and contact surfacealong their connecting loops.

Comparison of TBS-2 of the aligned molecules, reveals more subtleeffects. Phe⁶⁵⁰, which protrudes out into solvent in the GelA-CTD, aswell as Tyr⁶³⁶, Gly⁶⁵ 1 and Asp⁶⁵⁶ are not conserved in ClI-Ctd. Again,these changes create both a different contact surface and differentsurface potentials which would reduce the possibility of ClI-Ctd bindingTIMP-2 at this region.

Comparison of Related Sequences

A sequence alignment of other MMP C-terminal domains was performed (FIG.5) to see if features noted in the comparison of ClI-Ctd and GelA-CTDheld true for other MMP family members particularly those not known tobind TIMP-2. One of the most striking features of the alignment is howwell conserved some of the residues necessary for full TIMP-2 bindingare throughout many members of the MMP family. Just as in the comparisonwith ClI-Ctd, Lys⁵⁷⁹, Arg⁵⁹⁰, Lys⁶⁰⁴ and Lys⁶⁴⁵ are well conserved inmany members of the family. GelB-CTD shows the least homology among thisgroup of positively charged residues. Also, the negative charges inClI-Ctd, which occurred at Trp⁵⁷⁴, Lys⁵⁷⁶, and Ala⁶⁰⁹ in GelA-CTD arealso seen in many of the members of the MMP family. Only GelA, GelB andMT1-MMP do not place negative charges in the cavity. Further examinationof the sequence alignment shows that GelA-CTD has very little homologywith other member in the region between Ala⁶⁰⁹-Pro⁶¹⁴. These residuesmake up the loop region which connect blades III and IV. Other membersshow a lot of homology over the region and fit well to a DFPGIX (where Xis either G, D, E or P) consensus sequence. It is interesting to notethat GelA is only homologous in this region at Ile⁶¹³ whose sidechainsis buried in the structure and could not interact directly with boundTIMP-2.

The alignment of residues from the TBS-2 region shows that GelA and GelBare most similar, although not identical, over this stretch. Many ofthese residues, except for Leu⁶⁴⁵ and Lys⁶⁴⁶, make up most of what isconsidered TBS-2 in GelA-CTD. Asp⁶¹⁵ is also considered part of TBS-2and is homologous in GelB. MT1-MMP and stromelysin-3 are the next mostsimilar with residues which are identical to or make conservativesubstitutions at Asp⁶¹⁵ and Asp⁶⁵⁶.

Comparison of GelA-CTD and GelB-CTD

A comparison of aligned structures made between GelA-CTD and the modelof GClB-CTD shows they share more homology over the TIMP-2 bindingsurface than ClI-Ctd. As seen from the sequence alignment, residues inTBS-2 were highly homologous. Tyr⁶³⁶, Val⁶⁴⁸, Gly⁶⁵¹, Asp⁶¹⁵ and Asp⁶⁵⁶from GelA-CTD are structurally conserved in GelB-CTD. Only one residueis significantly different, Phe⁶⁵⁰ becomes Val⁶⁹⁴ in GelB-CTD. The turnconnecting the third and fourth strands of blade IV required rebuildingin GelB-CTD due to the insertion of residues. But for the most part,these residues were arranged similarly in both structures. The loopconnecting the third and fourth strand of blade IV had to be rebuilt toaccommodate the insertion of two residues. This increased the size ofthe loop, but still placed Leu⁶⁸⁸ and Asn⁶⁸⁹ of GelB-CTD near Leu⁶⁴⁵ andLys⁶⁴⁶ of GelA-CTD. So while no new charges are introduced, the contactsurface in this region would be somewhat different in GelB-CTD.

In contrast to TBS-2, TBS-1 of the model of GelB-CTD divergesdramatically from GelA-CTD. A great number of changes have been made inthe non-polar cavity residues. Trp⁵⁷⁴, Tyr⁵⁸¹, Phe⁵⁸⁸, Phe⁶⁰², andTrp⁶¹⁰ are not conserved in GelB-CTD. The sequence changes make thecavity much deeper in GelB-CTD with a cavity floor defined by thecontribution of non-polar atoms from Leu688 and Met⁶⁵³. Other residuesconserved between the two in TBS-1 are some of the positively chargedresidues which lie about the cavity. Lys⁵⁷⁹ and Arg⁵⁹⁰ of GelA-CTD areconserved in GelB-CTD. GelB-CTD makes a conservative substitution atLys⁵⁷⁶ where the positive charge is conserved. Other positive charges,such as Lys⁶⁰⁴ and Lys⁶⁴⁶ of GelA-CTD, become polar, but unchargedresidues in GelB-CTD. Overall, there are fewer positively chargedresidues in the TBS-1 region of GelB-CTD than found in either GelA-Ctdor ClI-Ctd. The loop region connecting blades III and IV in GelB-CTD,which shows intermediate homology to GleA-CTD, required slightrebuilding due to the insertion of Leu⁶⁵⁹ in GelB-CTD. The insertionmakes it impossible to model the C_(α) positions of the loop residuesidentically, so it is modeled to have a different structure than eitherGelA-CTD of ClI-Ctd. Pro⁶¹⁴ of GelA-CTD is conserved in GelB-CTD butdoes superimpose due to the rebuilding of the loop. Asn⁶¹¹ and Ala⁶¹²are different in GelB-CTD, but are identical to residues seen in theClI-Ctd structure.

Mutants of GelA-CTD That Don't Inhibit Membrane Dependant Activation ofGelA Are Clustered Within The TIMP-2 Binding Site.

Interaction of the GelA-CTD with cell surface is essential foractivation of the pro-enzyme. Consequently membrane dependent activationof GelA is competitively inhibited in the presence of the recombinantGelA-CTD (see introduction and discussion). The results we have reportedearlier support the hypothesis that assembly of MMP/TIMP-2/GelA-CTDcomplex promotes activation of GelA and inhibition of GelA activation inthe presence of excess of GelA-CTD is due to a direct competition withthe binding of GelA to the inhibitor TIMP-2 in the complex. A directapproach to the question whether the assembly of this complex is indeeda prerequisite for GelA activation is to determine whether activationinhibition and TIMP-2 binding properties of GelA-CTD can be separated.Therefore we investigated the ability of all 50 GelA-CTD mutantsdescribed above to inhibit membrane dependent activation of GelA invitro. Increasing amounts of purified WT or mutant GelA-CTD protein wasadded to membrane GelA activation reaction and the amount of remainingproenzyme species, a measure of activation inhibition, was analyzed onzymograms. The results are presented in FIG. 6. Most noticeable, is thefact that point mutations outside of the TIMP-2 binding site haveinhibited GelA activation as did WT GelA-CTD (T2+Ai⁺ phenotype).Furthermore, the only point mutations which showed a loss in activationinhibition were those found in the TIMP-2 binding site described above.However, mutants that exhibited a dramatic loss of TIMP-2 bindingactivity (Ki/Kd>100) segregated into two groups. Mutants of Lys⁵⁷⁶,Arg⁵⁹⁰, and Trp⁵⁷⁴ completely failed to inhibit GelA activation (T2⁻Ai⁻phenotype). Mutants of Asp⁶¹⁵, and Lys⁶⁴⁶ were indistinguishable fromWT, while mutant Glu⁶⁴¹+Gly⁶⁵¹→Arg shown only a slight loss ofactivation inhibition activity. Mutants Asp⁶⁵⁶ and Tyr⁶³⁶ exhibited asignificant loss of TIMP-2 binding (Ki/Kd=10) and a comparable loss ofactivation inhibition activity. Mutant Lys⁶⁰⁴ showed a considerable lossin TIMP-2 binding (Ki/Kd=25) but had little or no effect on activationinhibition. All other mutants (see table 1 and FIG. 6) characterized bya very moderate loss of TIMP-2 binding (Ki/Kd<10) and wereindistinguishable from WT in the activation inhibition assay. Thus pointmutants of residues in the TIMP-2 binding site do not always show acomplete correlation between the degree of loss of TIM P-2 binding andtheir respective loss of activation inhibition activity. Mutants that doshow such correlation are distributed between TBS1 and 2. Those withsevere loss of both functions (Trp⁵⁷⁴, Lys⁵⁷⁶, and Arg590) are clusteredtogether in the TBS-1 region of the TIMP-2 binding site (see FIG. 1).Two mutants with moderate effect on both functions (Asp⁶⁵⁶ and Tyr⁶³⁶)are found in TBS2. Two mutants with the greatest disparity in effect onTIMP-2 binding and activation inhibition (Asp⁶¹⁵ and Lys⁶⁴⁶) are foundon the border between TBS1 and 2. Finally it is important to note anabsence of the mutants with T2b⁺Ai⁻ phenotype.

Discussion.

Since GelA-CITD displays pseudo four-fold symmetry, it is interesting toconsider what structural features distinguish the TIMP-2 binding sitelocated roughly at the interface between blades III and IV from similarsites which would be found at the interfaces between the three otherblades. A GRASP representation of the GelA-CTD structure withelectrostatic potentials displayed at the surface of the molecule showsthat the interface between blades III and IV is unique in having a highconcentration of positive charge (FIG. 3) located near the interface.Furthermore, the outermost strand of blade IV is unique in the GelA-CTDstructure in that it forms a regular anti-parallel β-strand with noβ-bulges as seen in blades II and III. The fourth strand of blade 1contains no β-bulges, but its backbone H-bonding pattern with the thirdstrand is significantly distorted by the presence of cis proline,Pro⁵⁰⁶. Cis prolines are identified in the fourth strands of all theblades except IV. Thus, the highly localized positive charge and acanonical β-strand conformation of an adjacent blade would, in part,create a unique binding surface which would not be found at relatedpositions of this highly symmetrical molecule.

Having defined a TIMP-2 binding site on GelA-CTD, it is possible to lookat known structures and sequences of related MMPs and develop an idea ofhow binding and specificity are achieved. The two basic assumptions insuch an analysis are that 1) all related MMP sequences adopt the samefold as described for GelA-CTD and ClI-Ctd and 2) TIMP-1 binds Gel B-Ctdin a manner comparable to the TIMP-2 binding of GelA-CTD. If these twoassumptions are true than some interesting observations on the nature ofTIMPs binding to MMPs may be credibly made and are discussed below.

1) The positively, charged residues in TBS-1 of GelA-CTD are requiredbut not sufficient for binding TIMP-2.

While the mutation studies show that these residues are clearly requiredfor full TIMP-2 binding activity, the fact that many of these chargedresidues are conserved in MMPs which are not known to bind TIMP-2suggests that the presence of these residues is not sufficient forcausing TIMP-2 binding. TIMP-2 has a negatively charged C-terminal tailsequence, EFLDIEDP, which when removed shows a reduced binding kineticsprofile similar to that of TIMP-1 (30). TIMP-1 does not have anegatively charged sequence at its C-terminus. Since electrostaticforces often effect long range interactions between molecules, thepositive charges may serve to draw the TIMP-2 molecule near the bindingsite of GelA-CTD prior to docking. Once bound, the electrostaticinteractions are maintained, but van der Waal forces predominate indirecting full, specific binding. It is possible that the negativecharges described in the TBS-1 region of other non-TIMP binding MMPsreduce the effect of the long range interaction and also minimize theelectrostatic interaction between the negatively charged TIMP-2 sequenceand the conserved positively charged residues of these MMPs. It is alsointeresting to note that Gel B, which specifically binds TIMP-1, has twofewer positively charged residues than GelA in the TIMP-2 bindingsurface. Perhaps, these two residues, Lys⁶⁰⁴ and Lys⁶⁴⁶, play a role inbinding the negatively charge tail of TIMP-2. Also, Lys⁵⁹⁵ and Lys⁵⁹⁷,which were not mutated in this study, but are near the binding site, mayinteract with the TIMP-2 tail. Lys⁵⁹⁷ is of particular interest since itis not conserved in any of the other MMPs.

2) Interaction With TBS-1 Is Likely To Contribute More Than TBS-2 toSpecificity of TIMP-2 Binding to GelA-CTD.

GelA-CTD and Gel B-CTD share considerable homology in the TBS-2 regionso specificity will most likely not be determined in that region.Presumably, TIMP-1 and TIMP-2 will bind the TBS-2 region similarly inboth molecules. The region of the TIMP-2 binding site that diverges themost between GelA and B are found in TBS-1. Here, Gel B is missing twopositively charged residues. Also, sequence analysis and modelcomparison show the two would have different non-polar cavities. The GelB cavity is deeper and broader than that of GelA. Furthermore, the loopAla⁶⁰⁹-Asp⁶¹⁵ connecting blades III and IV of GelA-CTD is different thanthat of Gel B-Ctd. The loop differs in both sequence and backbonestructure by virtue of an insertion of a Leu residue in the Gel Bsequence.

3) Van der Waal forces play a major role in TIMP-2 binding andspecificity.

The TIMP-2 binding site of GelA-CTD represents a broad surface which isconservatively estimated to cover just over 1000 Å² and is composedmainly of uncharged residues. Of the charged residues in the bindingsite, many are found in the C-terminal domains of non-TIMP binding MMPssuggesting that the presence of the charged residues alone is not enoughto account for binding. Likewise, the fact that GelA-CTD shares so manycharged residues in common with Gel B-Ctd suggests that specific bindingof TIMP-2 is not a result of simple electrostatic interactions. Mostlikely, the strength and specificity of the binding comes as much fromvan der Waal interactions as from electrostatic attraction. Biochemicalstudies have shown that TIMP-2 binding to GelA-CTD is sensitive to lowpH and ionic detergent but resistant to high salt (20,30). These resultssuggest that there is both a significant ionic and van der Waalcomponent to the TIMP-2 binding of GelA-CTD. The TIMP-2 binding site ofGelA-CTD described in this paper represents a broad surface ofapproximately 1000 Å² with a high positively charged region clusteredabout a hydrophobic cavity and an extended, mostly uncharged, van derWaal contact surface. The charged region of the site accounts for the pHand ionic strength dependence of binding, while the cavity and broad,van der Waal surface of the site accounts for the requirement ofdetergent to fully disassociate the complex.

One of the most prominent sequence characteristics of non-TIMP bindingMMPs is their propensity to have negatively charged residues in or nearthe cavity in TBS-1. These charges were seen as potentially having adetrimental effect on TIMP-2 binding. As noted earlier, besides GelA andB, only MT1-MMP is identified as not having negative charges at residuesfound in or near the cavity. Furthermore, as seen in FIG. 4, many of thesequence features shared by GelA and B are also found in MT1-MMP.Pro⁶¹⁴, Asp⁶¹⁵, and Asp⁶⁵⁶ residues of GelA are conserved in MT1-MMP aswell. While there are still many sequence features among the TIMP-2binding site residues not shared by GelA and MT1-MMP, MT1-MMP is by farthe most homologous of the non-Gelatinase MMPs. Taken together, theseobservations suggest that MT1-MMP may be able to bind TIMP-2. In factrecent observations support this conclusion (25,26).

Interaction of inhibitors with pro-gelatinases is mediated by itsC-terminal domain (20-22). The TIMP-2 C-terminal domain is 67 residueslong from Cys¹²⁸-Pro¹⁹⁴. It has six cysteines which, by analogy toTIMP-1, are assumed to form three disulfide bonds (31). Thus, theC-terminal domain of TIMP-2 is likely to be compact and globular. TheC-terminal portion is separated from the N-terminal-domain by only asingle residue, Glu¹²⁷, so the N- and C-terminal domains of TIMP-2 mustbe located extremely close to one another in space. Given the largesurface area of the TIMP-2 binding site of GelA-CTD, it is possible thatportions of the N-terminal domain of TIMP-2 also participate in binding.Since the N-terminal domains of TIMP-1 and TIMP-2 show greater homology(44% identity) than their respective C-terminal domains (27% identity)and the TBS-2 sections of GelA and B are far more similar than theirTBS-1 regions, it is possible that portions of the N-terminal domain ofTIMP-2 binds blade IV residues of Gel-Ctd. This would mean that TBS-1 ofGelA-CTD is bound by the C-terminal portion of TIMP-2. As stated above,the C-terminal domain of TIMP-2 contains a negatively charged sequencewhich is required for full binding activity. TBS-1 has a lot ofpositively charged residues, particularly in GelA, and based on sequenceand model comparison of GelA and B shows far less sequence andstructural homology than in TBS-2. For this reason, it is likely thatTBS-1 of GelA determines its specificity for TIMP-2 as opposed toTIMP-1. Furthermore, assuming the C-terminal domain of TIMP-2 is notelongated, portions of TBS-2 may in fact bind parts of the N-terminaldomain of TIMP-2.

GelA is a multi-domain protein containing a catalytic domain, a domainwith thrice type II fibronectin-like repeats, and a C-terminal domain.The quaternary arrangement of these domains is still unknown.Biochemical evidence from deletion studies and crosslinking experimentssuggest that active GelA is bound simultaneously at its catalytic andC-terminal domains by the N-terminal and C-terminal domains of TIMP-2.TIMP-2 is a relatively small, globular protein (MW=21 kDa) whoseN-terminal portion is compact, adopts an OB-fold (32), and competitivelyinhibits substrate cleavage by binding the catalytic domain of MMPs.Given the TIMP-2 binding site described in the paper, it may be assumedthat the active site of the catalytic domain is located relatively nearthe interface between blades III and IV of the C-terminal domain whenbound to TIMP-2. Whether the domains of GelA adopt a rigid conformationor tumble freely in solution has yet to be determined and is the subjectof future study.

Mechanism of Cell surface GelA Activation.

The soluble MMP, GelA, is recruited to the cell surface where it isactivated in a MT1-MMP dependent fashion (reviewed 33). The initialMT1-MMP dependent Asn³⁷-Leu pro-peptide cleavage is inhibited by excessof TIMP-2 and competitively inhibited by GelA-CTD. Accordingly,truncated GelA that lacks its C-terminal domain is not activatable bythis mechanism (13). Thus compelling evidence supports the role ofGelA-CTD in recruitment of the proenzyme to the cell surface that is aprerequisite to its activation. The role of TIMP-2 in this mechanism ismore controversial. It is clear that the recombinant GelA-CTD caninteract with cell surface via binding to the activated MT1-MMP/TIMP-2complex to form a tri-molecular complex of activatedMT1-MMP/TIMP-2/GelA-CTD. It is also possible to demonstrate thatcarefully titrated amounts of TIMP-2 can increase the efficiency ofactivation in cell membrane dependent, TIMP-2 depleted system. Theseresults support the hypothesis that assembly of MT1-MMP/TIMP-2/GelA-CTDcomplex promotes cell surface GelA activation. Conversely, it has becomeevident that soluble MT1-MMP lacking its transmembrane domain canfaithfully cleave GelA propeptide at Asn³⁷-Leu (26). In this solublepurified system, TIMP-2 functions solely as a specific MT1-MMPinhibitor. Cleavage of the GelA propeptide does not depend on thepresence of its C-terminal domain and, contrary to membrane dependentGelA activation, truncated GelA is a substrate for soluble activatedMT1-MMP. Thus it is essential to ascertain by other approaches whetherthe assembly of the MT1-MMP/TIMP-2/GelA complex on the cell membrane isindeed a prerequisite for GelA activation.

Since inhibition of GelA activation in the presence of excess ofGelA-CTD is due to a direct competition with the cell surface binding ofGelA, a powerful approach to the above question is to determine whetheractivation inhibition and TIMP-2 binding properties of GelA-CTD can beseparated. Our previous experiments using chemical and protcolyticmodifications of GelA-CTD failed to achieve such an effect (9,10). Allmanipulations of GelA-CTD abolished both its TIMP-2 binding and theinhibitory activity in the membrane activation assay. Mutagenesisprovides an infinitely better approach to address this question. Acomplete correlation between loss of activation inhibition function andthe ability to bind TIMP-2 can be a conclusive evidence that TIMP-2serves as a mediator of GelA activation, provided that a sufficientnumber of the TIMP-2 binding site mutants were analyzed. Here, weinvestigate the ability of all fifty GelA-CTD mutants described above toinhibit membrane dependent activation of GelA in vitro. All mutantsoutside of the TIMP-2 binding site inhibit GelA activation as well as WTGelA-CTD (T2⁺Ai⁺ phenotype). Mutants that exhibited a dramatic loss ofTIMP-2 binding activity (Ki/Kd>100) segregated into classes. Mutantswith alanine substitution of Lys⁵⁷⁶, Arg⁵⁹⁰, and Trp⁵⁷⁴ failed toinhibit GelA activation (T2b⁻Ai⁻ phenotype). Asp⁶¹⁵, and Lys⁶⁴⁶ mutantswere indistinguishable from WT, while a double with alanine substitutingfor Glu⁶⁴¹ and Arg for Gly⁶⁵¹ shown only a slight loss of activationinhibition activity. Other mutants in the TIMP-2 binding site showmoderate to no effect on activation inhibition.

Importantly no mutants with T2⁺Ai⁻ phenotype were found. Thus, althoughall Ai⁻ mutants are concentrated in the TIMP-2 binding site, and noT2b⁺Ai⁻ mutants were isolated, the correlation between loss of TIMP-2binding and activation inhibition properties of GelA-CTD mutants is notabsolute. This inconsistency can be explained by differences in theassays used to measure the effects of the point mutations. For example,only a part of the TIMP-2 binding site of GelA-CTD described hereactually interacts with TIMP-2 bound to MT1-MMP. This may be due to thenature of TIMP-2 interaction with MT1-MMP that exposes only a portion ofTIMP-2 C-terminal domain necessary to engage the entire GelA-CTD bindingsite. Thus only a fraction of mutants in the GelA-CTD TIMP-2 bindingsite loses the capacity to competitively inhibit activation (T2⁻Ai⁻). Inthis case the assembly of MTl-MMP/TIMP-2/GelA complex is still aprerequisite for GelA activation and the question remains how theMT1-MMP occupied and inhibited by TIMP-2 is able to cleave the GelApropeptide. Several explanations can be invoked for the mechanism ofthis reaction. An activation model can be proposed where MT1-MMP/TIMP-2complex acts as a receptor for soluble GelA and forms a tri-molecularpresentation complex. Another molecule of TIMP-2 free MT1-MMP may thenperform the Asn³⁷-Leu pro-peptide cleavage. As a result, activation ofGelA is sensitive to the ratio of the unoccupied activated MT1-MMP toMT1-MMP/TIMP-2 complex and saturating amounts of TIMP-2 inhibitactivation.

A second set of GelA activation models can be proposed based on the datapresented here, if the existence of T2⁻Ai⁻ and T2⁻Ai⁺ mutants isinterpreted to mean that GelA-CTD binds to another, yet to beidentified, cell surface receptor and the resulting complex is activatedby TIMP-2 free MT1-MMP. For example, binding of the GelA-CTD can occurthrough interaction with α Vβ5 integrin as recently reported (34). Theresults of mutagenesis indicate that TIMP-2 and putative receptorbinding sites on GelA-CTD overlap since no Ai⁻ mutants were foundoutside of the TIMP-2 binding site. This overlap can potentially explainwhy TIMP-2 can inhibit binding of GelA to the cell surface even in thecase that it is mediated by a receptor other than MT1-MMP/TIMP-2complex. Earlier we have described an analogous but soluble complex ofGelB/ClI where the ClI and TIMP-1 binding sites of GelB-CTD overlap(22).

TABLE 1 Gel A-CTD mutants that affect its TIMP-2 binding activity(Ki/Kd > 1). Wild type Kd and mutant Ki was determined as in FIG. 2. Dand ID - mark mutants affecting TIMP-2 binding directly and indirectlyrespectively. Mutant # Ki/Kd Mutant # Ki/Kd #28 ID 8 #247 D >500Asp^(569→)Ala Lys^(646→)Ala #31 D 6 #250 D >500 Lys^(579→)AlaTrp^(574→)Ala #39 D 25 #252 D 10 Lys^(604→)Ala Tyr^(636→)Ala #41 D 300#255 D 8 Asp^(615→)Ala Phc^(650→)Ala #46 ID >500 #257 D 3 Glu^(641 →)Ala + Gly^(651→)Ala Gly^(651 →) Arg #229 D ≧500 #258 ID 10 Lys^(576→)AlaAsp^(656→)Ala #234 D >500 #259 D 3 Arg^(590→)Ala Asn^(611→)Ala

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SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 19(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Asn TrpSer Lys Asn Lys Lys Thr Tyr Ile Phe Ala Gly Asp Lys 5 10 15 Phe Trp ArgTyr Asn Glu Val Lys Lys Lys Met Asp Pro Gly Phe 20 25 30 Pro Lys Leu IleAla Asp Ala Trp Asn Ala Ile Pro 35 40 (2) INFORMATION FOR SEQ ID NO: 2:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: aminoacid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID:NO: 2: Arg Ser Gly Arg Gly Lys Met Leu Leu Phe SerGly Arg Arg Leu 5 10 15 Trp Arg Phe Asp Val Lys Ala Gln Met Val Asp ProArg Ser Ala 20 25 30 Ser Glu Val Asp Arg Met Phe Pro Gly Val Pro Leu 3540 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Phe GluGlu Asp Thr Gly Lys Thr Tyr Phe Phe Val Ala His Glu 5 10 15 Cys Trp ArgTyr Asp Glu Tyr Lys Gln Ser Met Asp Thr Gly Tyr 20 25 30 Pro Lys Met IleAla Glu Glu Phe Pro Gly Ile Gly 35 40 (2) INFORMATION FOR SEQ ID NO: 4:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: aminoacid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 4: Ser Glu Glu Asn Thr Gly Lys Thr Tyr Phe PheVal Ala Asn Lys 5 10 15 Tyr Trp Arg Tyr Asp Glu Tyr Lys Arg Ser Met AspPro Ser Tyr 20 25 30 Pro Lys Met Ile Ala His Asp Phe Pro Gly Ile Gly 3540 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: His PheGlu Asp Thr Gly Lys Thr Leu Leu Phe Ser Gly Asn Gln 5 10 15 Val Trp ArgTyr Asp Asp Thr Asn His Ile Met Asp Lys Asp Tyr 20 25 30 Pro Arg Leu IleGlu Glu Asp Phe Pro Gly Ile Gly 35 40 (2) INFORMATION FOR SEQ ID NO: 6:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (B) TYPE: aminoacid (C) TOPOLOGY: linear (II) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 6: Ser Asp Lys Glu Lys Asn Lys Thr Tyr Phe PheVal Glu Asp Lys 5 10 15 Tyr Trp Arg Phe Asp Glu Lys Arg Asn Ser Met GluPro Gly Pro 20 25 30 Lys Gln Ile Ala Glu Asp Phe Pro Gly Ile Asp 35 40(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 42 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Ser AspLys Glu Lys Lys Lys Thr Tyr Phe Phe Ala Ala Asp Lys 5 10 15 Tyr Trp ArgPhe Asp Glu Asn Ser Gln Ser Met Glu Gln Gly Phe 20 25 30 Pro Arg Leu IleAla Asp Asp Phe Pro Gly Val Glu 35 40 (2) INFORMATION FOR SEQ ID NO: 8:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: aminoacid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 8: Trp Gly Pro Glu Lys Asn Lys Ile Tyr Phe PheArg Gly Arg Asp 5 10 15 Tyr Trp Arg Phe His Pro Ser Thr Arg Arg Val AspSer Pro Val 20 25 30 Pro Arg Arg Ala Thr Asp Trp Arg Gly Val Pro Ser 3540 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 40 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Trp MetPro Asn Gly Lys Thr Tyr Phe Phe Arg Gly Asn Lys Tyr 5 10 15 Tyr Arg PheAsn Glu Glu Leu Arg Ala Val Asp Ser Glu Tyr Pro 20 25 30 Lys Asn Ile LysVal Trp Glu Gly Ile Pro 35 40 (2) INFORMATION FOR SEQ ID NO: 10: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 (B) TYPE: amino acid (C)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 10: Asp Asn Leu Asp Ala Val Val Asp Leu Gln Gly Gly Gly HisSer 5 10 15 Tyr Phe Phe Lys Glu Ala Tyr Tyr Leu Lys Leu Glu Asn Gln Ser20 25 30 Leu Lys Ser Val Lys Phe Gly Ser Ile Lys Ser Asp Trp Leu Gly 3540 45 Cys (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 11: Asp Thr His Asp Val Phe Gln Tyr Arg Glu Lys Ala Tyr PheCys 5 10 15 Gln Asp Arg Phe Tyr Trp Arg Val Ser Ser Arg Ser Glu Leu Asn20 25 30 Gln Val Asp Gln Val Gly Tyr Val Thr Tyr Asp Ile Leu Gln Cys 3540 45 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 43 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:Asn Lys Val Asp Ala Val Phe Gln Lys Asp Gly Phe Leu Tyr Phe 5 10 15 PheHis Gly Thr Arg Gln Tyr Gln Phe Asp Phe Lys Thr Lys Arg 20 25 30 Ile LeuThr Leu Gln Lys Ala Asn Ser Trp Phe Asn Cys 35 40 (2) INFORMATION FORSEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids(B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: His Lys Val Asp Ala Val PheMet Lys Asp Gly Phe Phe Tyr Phe 5 10 15 Phe His Gly Thr Arg Gln Tyr LysPhe Asp Pro Lys Thr Lys Arg 20 25 30 Ile Ile Thr Leu Gln Lys Ala Asn SerTrp Phe Asn Cys 35 40 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (C)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 14: Asp Lys Val Asp Ala Val Tyr Glu Lys Asn Gly Tyr Ile TyrPhe 5 10 15 Phe Asn Gly Pro Ile Gln Phe Glu Tyr Ser Ile Trp Ser Asn Arg20 25 30 Ile Val Arg Val Met Pro Ala Asn Ser Ile Leu Trp Cys 35 40 (2)INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:43 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Ser Lys Ile AspAla Val Phe Glu Glu Phe Gly Phe Phe Tyr Phe 5 10 15 Phe Thr Gly Ser SerGln Leu Glu Phe Asp Pro Asn Ala Lys Lys 20 25 30 Val Thr His Thr Leu LysSer Asn Ser Trp Leu Asn Cys 35 40 (2) INFORMATION FOR SEQ ID NO: 16: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: aminoacid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 16: Pro Lys Val Asp Ala Val Leu Gln Ala Phe GlyPhe Phe Tyr Phe 5 10 15 Phe Ser Gly Ser Ser Gln Phe Glu Phe Asp Pro AsnAla Arg Met 20 25 30 Val Thr His Ile Leu Lys Ser Asn Ser Trp Leu His Cys35 40 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 46 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:Glu Ile Asp Ala Ala Phe Gln Asp Ala Asp Gly Tyr Ala Tyr Phe 5 10 15 LeuArg Gly Arg Leu Tyr Trp Lys Phe Asp Pro Val Lys Val Lys 20 25 30 Ala LeuGlu Gly Phe Pro Arg Leu Val Gly Pro Asp Phe Phe Gly 35 40 45 Cys (2)INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:48 amino acids (B) TYPE: amino acid (C) TOPOLOGY:linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Glu Ser Pro ArgGly Ser Phe Met Gly Ser Asp Glu Val Phe Thr 5 10 15 Tyr Phe Tyr Lys GluAsn Lys Tyr Trp Lys Phe Asn Asn Gln Lys 20 25 30 Leu Lys Val Glu Pro GlyTyr Pro Lys Ser Ala Leu Arg Asp Trp 35 40 45 Met Gly Cys (2) INFORMATIONFOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 83 aminoacids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE:peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: Trp Ser Lys Asn LysLys Thr Tyr Ile Phe Ala Gly Asp Lys Phe 5 10 15 Trp Arg Tyr Asn Glu ValLys Lys Lys Met Asp Pro Gly Phe Pro 20 25 30 Lys Leu Ile Ala Asp Ala TrpAsn Ala Ile Pro Asp Asn Leu Asp 35 40 45 Ala Val Val Asp Leu Gln Gly GlyGly His Ser Tyr Phe Phe Lys 50 55 60 Glu Ala Tyr Tyr Leu Lys Leu Glu AsnGln Ser Leu Lys Ser Val 65 70 75 Lys Phe Gly Ser Ile Lys Ser Asp 80

What is claimed is:
 1. A target area for screening test compounds asinhibitors of matrix metalloproteinases consisting of a peptide ofresidues Trp⁵⁷⁴ to Asp⁶⁵⁶ in the tissue inhibitor of 72 kDa type IVcollagenase binding site of the C-terminal domain of gelatinase-A asshown in SEQ ID NO:19.
 2. A method of screening a test compound forinhibition of matrix metalloproteineases comprising: a) contacting saidtest compound and said metalloproteinease, and b) determining theinhibitory effect of said test compound in a competitive inhibitionassay in which displacement of ¹²⁵I-labeled peptide bound to the tissueinhibitor of 72 kDa type IV collagenase by unlabeled test compound andunlabeled peptide is measured and the K_(i)/K_(d) ratio is determined;wherein K_(i)/K_(d)≧1 indicates inhibition, K_(i) is the inhibitoryconstant of said test compound, K_(d) is the dissociation constant ofsaid peptide, and wherein said peptide consists of residues Trp⁵⁷⁴ toAsp⁶⁵⁶ in the tissue inhibitor of 72 kDa type IV collagenase bindingsite of the C-terminal domain of gelatinase A as shown in SEQ ID NO. 19.